Gene Cloning Of Rice Yellow-green Mutant Ys83 And Brittle Mutant B364

Characterization of variety rice(Oryza sativa L.) mutants on genetic and molecular level through forward or reverse genetic screen methods revealed many essential gene functions and biological processes in higher plants, which contributed to theoretical and application value. Rice leaf-color mutants and brittle mutants are considered common research materials. Research on rice leaf-color mutants and genes provided theoretical basis for discovering cell processes such as biosynthesis of plant photosynthetic pigments, chloroplast development and nuclear-plastid signaling. Exploration on brittle mutants and genes also opened new thoughts on biosynthesis of cell wall, synthesis and degradation of cellulose even development of biomass energy. Our group obtained a yellow-green mutant ys83 and a brittle mutant b364 through chemical mutagenesis. Through phenotype characterization, map-based cloning and gene functional analysis, these two mutants are systematically and deeply studied, and the main contents are summarized as follows:1. Gene cloning and functional analysis of rice yellow-green mutant ys83 ys83 mutant exhibites a yellow-green phenotype during the seedling stage, while slowly turns into light-green leaves as it grows. At the mature stage, ys83 still showed pale green leaves that could be distinguished from the wild-type plant. Regarding to the main agronomic traits of mature stage, number of productive panicles per plant and number of spikelets per panicle of ys83 reduce by 6.7% and 7.6% compared with those of wild-type parent Nipponbare, respectively. However, agronomic traits on other aspects are not affected significantly in ys83. Measurement and comparison on photosynthetic pigments in ys83 mutant and wild type plant reveal that, contents of chlorophyll and carotenoids during seedling and heading stages of ys83 mutants are severely reduced, especially pigments contents of mutant seedlings which are only a half of that of wild type plant. This result indicates that the yellow-green leaf phenotype of ys83 mutant is caused by reduction of chlorophyll. Compared with wild type plant, chloroplast development is retarded in ys83 mutant, with less and thinner grana stacks. Through map-base cloning method, the ys83 locus is located in a 158kb region on the short arm of rice Chr.2. There are 23 annotated genes depending on the rice genome annotation project. After PCR amplification and sequencing analysis, gene LOC_Os02g05890 is considered as the candidate gene of ys83 mutant, which is named as YS83. A T-A substitution at position 198 of YS83 cDNA region occurs, turning the 66th codon TGT to TGA and resulting in a premature translation of the encoding protein. Furthermore, complementation experiment is performed to confirm this result. The full length of cDNA sequence of YS83 from NP is ligated into vector pCAMBIA2300 and the resulting plasmid pC2300-YS83 is transformed into ys83 mutants. Transgenic plants possessing positive PCR bands recover to normal green phenotype and same content of pigments as wild type plants. These results demonstrate that YS83 is the corresponding gene of the mutant phenotype of ys83.YS83 is a single-copy gene in rice with a 498-bp ORF, encoding a 165-amino acid protein with a molecular weight of 17 kDa. The YS83 protein contains a predicted chloroplast transit peptide of 51 amino acids towards the N terminus and a putative functional glycine-rich domain close to the C terminus. Further research on subcellular localization experiments confirms that protein YS83 is targeted to the chloroplast. Multiple alignments and phylogenetic tree of YS83 and its homologous proteins reveal that rice YS83 has higher similarity and closer relationship to its homologues of monocotyledonous plants than to those of dicotyledons plants.Gene YS83 is expressed ubiquitously in a wide range of tissues, while higher transcription activity is found in vigorous growth tissues. Compared with wild type plant, expression level of several genes related to chloroplast development and chlorophyll synthesis are affected differently in ys83 mutant, implying that YS83 could be required for normal development of chloroplast through regulation essential genes related to chloroplast biogenesis and chlorophyll biosynthesis in rice.2. Genetic analysis and gene cloning of rice brittle mutant b364The whole plant of brittle mutant b364 is brittle, including its root, culm, leaf and spike, especially leaf and culm. However except brittleness, other morphological phenotype and field traits are no different from wild type plant. When observed under environmental scanning electron microscope, sclerenchyma structure of wild type plant is much tighter, while cell arrangement of b364 mutant is looser and the structure is hollow. Then, cell wall structure is observed using transmission electron microscope. Cell wall thickness of leaf vein cells in b364 mutant is obviously thinner than that in wild type plant, which has no typical structure of three components, and change in secondary cell wall is most serious. These results imply that cell wall biosynthesis is blocked in b364 mutant, resulting in reducing mechanical strength, especially the secondary cell wall of sclerenchyma cells which providing the main mechanical strength for plants.All of the F1 plants obtained from crossing of b364 with the wild type parent NP, indica cv. Minghui63 and G46B, exhibit brittle phenotype, indicating the brittle phenotype is dominant. The F2 population shows a 3:1 ratio on number of brittle plants and normal plants, which implies that the brittle phenotype of b364 is controlled by a pair of dominant nuclear genes. Further, b364 locus is located in a DNA region of 145kb on the long arm of rice Chr.l using map-based cloning. There are 23 annotated genes in this region, of which 8 are transposons. Depending on the annotated gene functions of the rest 15 genes,4 genes are preferentially selected as candidates and are amplified and sequenced. Then, one gene is found a single basepair mutation in it which causes an amino acid change in its encoding protein, so this gene is considered as the candidate gene of brittle phenotype of b364 and is named as B364. Dominant mutant gene b364 is transformed into wild type plants by transgenic technology, and positive transgenic plants exhibit brittle phenotype, which further verify this candidate gene. These data reveal that the brittle feature of b364 is caused by mutation in gene B364.